Bottom Line:
Primary cilia are nonmotile microtubule structures that assemble from basal bodies by a process called intraflagellar transport (IFT) and are associated with several human diseases.Pcnt is found in spermatocyte IFT fractions, and IFT proteins are found in isolated centrosome fractions.We conclude that Pcnt, IFTs, and PC2 form a complex in vertebrate cells that is required for assembly of primary cilia and possibly motile cilia and flagella.

ABSTRACTPrimary cilia are nonmotile microtubule structures that assemble from basal bodies by a process called intraflagellar transport (IFT) and are associated with several human diseases. Here, we show that the centrosome protein pericentrin (Pcnt) colocalizes with IFT proteins to the base of primary and motile cilia. Immunogold electron microscopy demonstrates that Pcnt is on or near basal bodies at the base of cilia. Pcnt depletion by RNA interference disrupts basal body localization of IFT proteins and the cation channel polycystin-2 (PC2), and inhibits primary cilia assembly in human epithelial cells. Conversely, silencing of IFT20 mislocalizes Pcnt from basal bodies and inhibits primary cilia assembly. Pcnt is found in spermatocyte IFT fractions, and IFT proteins are found in isolated centrosome fractions. Pcnt antibodies coimmunoprecipitate IFT proteins and PC2 from several cell lines and tissues. We conclude that Pcnt, IFTs, and PC2 form a complex in vertebrate cells that is required for assembly of primary cilia and possibly motile cilia and flagella.

Mentions:
Because vertebrate primary cilia formation and function requires IFT proteins (Murcia et al., 2000; Pazour et al., 2000) and the cation channel PC2 (Somlo and Ehrlich, 2001; Pazour et al., 2002b; Rosenbaum and Witman, 2002; Nauli et al., 2003), we reasoned that Pcnt might cooperate with these proteins in primary cilia organization. To test this, we first determined the precise localization of these proteins. IFT57 and IFT88 localized primarily to the distal end of the mother centriole near the base of the primary cilium and to the tips and in spots along the length of primary cilia (Fig. 3, a and b). Localization of these IFTs to the distal portion of the mother centriole was consistent with known sites of IFT protein localization in Chlamydomonas reinhardtii (Cole et al., 1998; Deane et al., 2001). IFT20 was found on the proximal portion of mother centriole and the lateral aspect of the daughter centriole (Fig. 3 c), an area thought to be involved in interconnecting the two centrioles. PC2 localized primarily to the mother centriole underlying the primary cilium (Fig. 3 d). In mouse tracheal epithelial cells, IFT proteins partially localized with Pcnt to sites at the base of the motile cilia where basal bodies are found (Fig. 3 e, IFT20).

Mentions:
Because vertebrate primary cilia formation and function requires IFT proteins (Murcia et al., 2000; Pazour et al., 2000) and the cation channel PC2 (Somlo and Ehrlich, 2001; Pazour et al., 2002b; Rosenbaum and Witman, 2002; Nauli et al., 2003), we reasoned that Pcnt might cooperate with these proteins in primary cilia organization. To test this, we first determined the precise localization of these proteins. IFT57 and IFT88 localized primarily to the distal end of the mother centriole near the base of the primary cilium and to the tips and in spots along the length of primary cilia (Fig. 3, a and b). Localization of these IFTs to the distal portion of the mother centriole was consistent with known sites of IFT protein localization in Chlamydomonas reinhardtii (Cole et al., 1998; Deane et al., 2001). IFT20 was found on the proximal portion of mother centriole and the lateral aspect of the daughter centriole (Fig. 3 c), an area thought to be involved in interconnecting the two centrioles. PC2 localized primarily to the mother centriole underlying the primary cilium (Fig. 3 d). In mouse tracheal epithelial cells, IFT proteins partially localized with Pcnt to sites at the base of the motile cilia where basal bodies are found (Fig. 3 e, IFT20).

Bottom Line:
Primary cilia are nonmotile microtubule structures that assemble from basal bodies by a process called intraflagellar transport (IFT) and are associated with several human diseases.Pcnt is found in spermatocyte IFT fractions, and IFT proteins are found in isolated centrosome fractions.We conclude that Pcnt, IFTs, and PC2 form a complex in vertebrate cells that is required for assembly of primary cilia and possibly motile cilia and flagella.

ABSTRACTPrimary cilia are nonmotile microtubule structures that assemble from basal bodies by a process called intraflagellar transport (IFT) and are associated with several human diseases. Here, we show that the centrosome protein pericentrin (Pcnt) colocalizes with IFT proteins to the base of primary and motile cilia. Immunogold electron microscopy demonstrates that Pcnt is on or near basal bodies at the base of cilia. Pcnt depletion by RNA interference disrupts basal body localization of IFT proteins and the cation channel polycystin-2 (PC2), and inhibits primary cilia assembly in human epithelial cells. Conversely, silencing of IFT20 mislocalizes Pcnt from basal bodies and inhibits primary cilia assembly. Pcnt is found in spermatocyte IFT fractions, and IFT proteins are found in isolated centrosome fractions. Pcnt antibodies coimmunoprecipitate IFT proteins and PC2 from several cell lines and tissues. We conclude that Pcnt, IFTs, and PC2 form a complex in vertebrate cells that is required for assembly of primary cilia and possibly motile cilia and flagella.